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Understanding the “Hayflick Limit”

Leonard Hayflick, 1961 at the Wistar Institute

I met with an eminent scientist in the course of my work, and as part of the background research, I checked out his CV (curriculum vitae). His background is substantial, Harvard education and NIH post-doc, but his mentor during his post-doc was Leonard Hayflick. I knew about the Hayflick Limit from grad school, but I had never read the history of the phenomenon. I thought it might make an interesting example of how science is conducted. We’ll come to a definition shortly.

You may be aware that scientists routinely grow human cells in flasks. It’s called cell culture or tissue culture. We’re not talking about an arm or an ear, here. The cells are only visible as a thin layer of translucent material, in the case of cells that adhere to a solid surface, or a slightly grainy slurry, in the case of cells in suspension. We feed these cells, and they are bathed in, a solution containing salts, sugars, amino acids and other essential nutrients. They also sometimes get antibiotics and growth stimulants. Most also receive a healthy dose of serum, extracted from cow blood, in order to provide specialized proteins that stimulate growth. We might say, for example, that our K-562 cells (immortalized cells from a patient with myelogenous leukemia) grow in RPMI1640 (a specific sugar/salt/amino acid solution) supplemented with 10% FBS (fetal bovine serum) plus pen/strep (penicillin/streptomycin) and L-glutamine (needed by some cells, and somewhat unstable in

Cell culture flasks with culture medium

storage). It looks complicated, but is not much different from a recipe for chicken soup. The flasks containing cells in their growth medium are placed in incubators, usually at 37 C and 5% CO2. Periodically, the medium has to be changed, and if the cells are rapidly growing, they have to be passaged or “split”, removing a fraction of the growing cells, to avoid overgrowth. We guard carefully against introducing infectious agents, since the cells have no immune system and any bacteria or fungus landing in the cell culture medium, loaded as it is with the materials of life, will rapidly overgrow, acidify the solution, and kill the cells.

So those are the very basics of growing mammalian cells in culture. Now we come to the interesting part. If I take a small scraping of your cheek and transfer them to a flask, they will grow for some time, then eventually they’ll stop growing and instead begin dissolving. The number of divisions that occur before this happens is called the Hayflick limit. Initially scientists assumed this was a technical problem with how they were culturing… not enough nutrients, or the introduction of bacteria or fungus pathogens… something was ending the life of the cells.

If they split the cells into two flasks, both cultures stopped replicating at about the same time, which made simultaneous infections unlikely.

Cells taken from embryonic tissues lasted much longer than mature cells taken from older patients. Hayflick measured the number of times an embryonic cell could divide. It converged on a value of about 50 divisions. This would later be called the Hayflick limit.

Many cancer cells (~80%) do not obey the Hayflick limit, they are "immortalized".

So once the phenomenon was known and the puzzle was well-characterized, a number of hypotheses were advanced to explain the anomalous observation. They focused on the exceptions to the Hayflick limit rules, cells that never stop growing, which we call immortalized cells. These cells were almost universally taken from either cells infected with specific viruses, or from very active cancer cells.

So what do these two conditions have in common? Both had damage to their DNA in certain specific regions that mediated cell division and self-destruct mechanisms. Most cells recognize their genetic damage as a good reason to self-destruct, to undergo apoptosis. These cells no longer had the ability to respond to that self-destruct signal. That alone wouldn’t have been enough, though. They had also acquired an enzyme that was able to add new sequences to the ends of their chromosomes. These sequences are called telomeres.

Telomeres are to the chromosome as the aglet (the little plastic or metal collar) is to the shoelace. The telomere prevents the DNA strand from “unraveling” by providing a long string of very tightly bound base pairs. One of the consequences of copying your DNA is that a little chunk from the very end of each of your 46 chromosomes is lost every time they are copied. When you are a fetus, you have nice long telomeres securing your chromosome ends. As you age, as the result of replicating your DNA, they get shorter. Eventually, they become too short to hold the ends together, and we think that’s why some cells must stop replicating.

Enter telomerase, a cellular enzyme that extends the length again. In my next post, I’ll examine what it does in the cell; whether it can be harnessed to make us essentially immortal, and we’ll examine some of the claims of futurists and anti-aging researchers. We’ll also look at why evolution favors our aging and eventual death, and those rare human genomes that have achieved near immortality.

I’ve just use something I learned on “Phineas and Ferb” in a science posting. Day complete.